Immunoglobulins Harry W Schroeder Jr MD PhD Division of Developmental and Clinical Immunology Departments of Medicine, Microbiology, and Genetics University of Alabama at Birmingham
Immunoglobulin Has Two Roles Antigen Receptor Recognition of and binding to antigens such as toxins, viruses, and exposed molecules on the surface of pathogenic organisms V domain function Effector Molecule Elimination or inactivation of the foreign antigen or the cell that bears the antigen C domain function
Immunoglobulins are Heterodimers Composed of Two H chains and Two L chains V J 5' 3' 5' C L L L N D J V C H H H H 1 Hinge C H 2 C H 3 3' N N S S S S S S S S Papain Pepsin Fc C S S S S S Gm H N 1 2 3 4 FR S S S S L N C 1 2 3 CDR Fab Km
Human Igκ Locus V domain is created by the joining of a Variable (V) and a Joining (J) gene segment Contains only one C gene, with only one exon IGKC: 1 IGKJ: 5 IGHV:40 Lefranc M.-P. Immunologist 8:80,2000
Human vs Mouse Human (2p11.2) Centromere Telomere J2 J4 J5 J3 3D-7 1D-43 3D-11 1D-12 3D-15 1D-16 1D-17 3D-20 2D-28 (2D-29) 2D-30 1D-33 1D-37 1D-39 2D-40 2-40 1-39 1-37 1-33 2-30 (2-29) 2-28 1-27 2-24 3-20 1-17 1-16 (1-13) 3-15 1-12 1D-8 C 3-11 1-9 1-5 1-6 5-2 4-1 J1 Mouse (6) 1-135 1-133 1-132 14-130 9-129 9-128 11-125 9-123 1-122 17-121 9-120 9-119 1-117 2-112 14-111 1-110 2-109 16-104 14-100 1-99 12-98 10-96 10-95 10-94 19-93 4-92 4-91 4-90 12-89 1-88 4-86 13-85 13-84 4-80 4-81 4-79 17-127 9-124 J1 J4 J5 J2 4-58 4-57 4-55 4-54 4-53 4-52 4-50 12-46 5-45 12-44 5-43 12-41 5-39 12-38 5-37 18-36 8-34 7-33 6-32 8-30 6-29 8-28 8-27 6-25 8-24 6-23 8-21 6-20 8-19 6-17 8-16 6-15 6-14 3-12 6-13 3-10 4-51 5-48 4-74 4-73 4-72 4-71 4-70 4-69 4-68 4-61 4-63 4-78 C 3-9 3-7 3-4 3-5 3-3 2-137 4-59 3-2 3-1
Creation of a κ chain 3-14 1-15 1-17 1-19 Vκ κ (40) 1-15 3-14 1-12 1-8 5-3 4-1 Jκ 1 2 3 4 5 Cκ Germline Jκ 1-15 3-14 1-12 1-8 2 1 4-1 5-3 3 4 5 Cκ Inversion Rearrangement Deletion + 1-8 5-3 4-1 1 Transcription L V J C 3 2 AAA L V J C mrna 5' AAA 3' Initial polypeptide L V J C Mature κ L chain V J C Vκ Cκ
Human Igλ Locus V domains composed of V and J segments Four or more equivalent single domain C genes, each with its own J IGHV:32 IGKJ: 4 IGJC: 4 Lefranc M.-P. Immunologist 8:80,2000
Human vs Mouse J1 C1 J3 C3 J7 C7 J2 C2 Human (22q11.2) Centromere Telomere 4-69 8-61 4-60 6-57 11-55 10-54 VpreB 5-52 1-51 9-49 5-48 1-47 7-46 5-45 1-44 7-43 1-40 (5-39) 5-37 1-36 3-27 3-25 2-23 3-22 3-21 3-19 2-18 3-16 2-14 3-12 2-11 3-10 3-9 4-3 2-8 3-1 λ5 V2 V3 J2 C2 V1 J3 C3 J1 C1 Mouse (16)
The Human IgH Locus Includes Diversity (D) as well as V and J gene segments Contains multiple, different C genes each with three or domains IGHC: 9 IGHJ: 6 IGHD:27 IGHV:39 Lefranc M.-P. Immunologist 8:80,2000
V11 V14 DDFL16.1 DSP2.3 DFL16.2 DSP2.4 DSP DSP DSP DSP DSP2.7 DSP2.8 DST4 DQ52 J1 J2 J3 J4 γ2b γ2a γ2 γ2 γ2 γ2 V15 V10 (DNA4) V12 V13 V11 V14 D1-1 D2-2 D3-3 D4-4 D5-5 D6-6 D1-7 D2-8 D3-9 D3-10 D4-11 D5-12 D6-13 D1-14 D2-15 D3-16 D4-17 D5-18 D6-19 D1-20 D3-21 D3-23 D4-23 D5-24 D6-25 D1-26 D7-27 J1 J2 J3 J4 J5 J6 3-74 3-73 3-72 2-70 1-69 3-66 3-64 (4-60) 4-59 1-58 3-53 5-51 3-49 3-48 1-46 1-45 3-43 4-39 4-34 (3-30.5) (4-30.4) (3-30.3) (4-30.2) (4-30.1) 3-30 4-28 2-26 1-24 3-23 3-21 3-20 3-19 1-18 3-15 3-13 (3-11) 2-10 3-9 1-8 3-7 2-5 (7-4.1) 4-4 1-3 1-2 6-1 Human (14q32.33) Mouse (12) Human vs Mouse Telomere Centromere µ δ γ3 γ3 γ1 γ1 α1 α1 γ2 γ2 γ4 γ4 ε α2 α2 V10 (DNA4) V9 (Vgam3.8) V1, V8 (>60) (J558, 3609) V6(~12) (J606) V3 (36-60) V7 (S107) V9 (Vgam3.8) V3 (3660) V3 (36-60) V4 (X24) V7 (S107) V2, V5 (~35-40) (Q52, 7183) µ δ γ1 γ3 ε α VH81x
Primary Structure - H chain V Domain
Non-Homologous End-Joining Site-specific cleavage RAG 1, RAG 2, HMG1, 2 Recognition of cut ends DNA PK Ku 70/80 Repair of the ends XRCC4 DNA ligase IV Artemis Modified repair TdT
Recombination Signal Sequences RSS - Conserved sequence flanking V (D) J gene segments Consists of a highly conserved seven bp sequence (heptamer), a less conserved nine bp sequence (nonamer), and a 12 or 23 bp (spacer) One-turn/two-turn rule (12/23 rule) Recombination occurs only between a 12 bp spacer RSS and a 23 bp spacer RSS H Chain VH H 23 N DH H 12 N 12 H N N 23 H JH κ Chain Vκ H 12 N N 23 H Jκ λ Chain Vλ H 23 N N 12 H Jκ
Rearrangement
TdT Adds nucleotides at random to the 3 terminus of DNA Expressed in fetal Expressed in fetal liver, bone marrow, and thymus
Junctional Diversity Hairpin Clip V GA CT TT AA D J Hairpin Clip V GATC TTAA D J Joining & Repair V P Junctions GATCAATT CTAGTTAA D J Nibbling, Joining & Repair V GT CA D J Nibbling, N addition Joining & Repair V N addition GGCATGGAT CCGTACCTA D J
Combinatorial Diversification H Chain V D J C C 23 12 12 23 39 x Deletion x 6 = 4 x 10 5 Inversion L Chain V 27 x 6 J x C C 23 12 40 x 5 = 2 x 10 2 = ~1 x 10 8
Junctional Diversification Combinatorial Diversity (V*D*J, V*J) = 1 x 10 8 CDR3 V 23 12 J x 1 x 10 1 Exonucleolytic Nibbling V D J x 23 12 12 23 Exonucleolytic Nibbling P Junctions N Nucleotide Addition 1 (2 x 10 ) per codon 6 codons x 1 x 10 7 = 1 x 10 16
A Nested Gradient of Diversity LIGHT CHAIN HEAVY CHAIN V H FR 1 2 3 D H J H C H FR4 HCDR3 HCDR1 LCDR2 CDR 1 2 FR3 FR3 (Family) V H N N J H C H FR3 C W FR4 C H 1 FR1 FR1 (Clan) CDR3 N - D - N H LCDR1 LCDR3 FR4 HCDR2
Class switching During an immune response, B cells can replace an upstream C domain with a downstream C domain class-switching B cells can thus produce IgG, IgA or IgE with the same antigen specificity as the original IgM
Class Switching Occurs by gene rearrangement Mediated by AID instead of RAG Involves non-homologous recombination between a pair of switch sites rather than pairs of RSS The switch sites are composed of multiple nucleotide repeats Transcription of the switch site is required Initiation of transcription is driven by T cell cytokines (e.g., IL-4 drives IgE and IgG4 in human) L V DJ Cµ Cδ C γ3 C γ1 C α1 C γ2 C γ4 C α2 Cε 5' 3' Sµ S γ3 S γ1 S α1 S γ2 S γ4 S α2 Sε
Recombination Occurs by Deletion Initial recombination occurs between Sµ and a downstream S site Allows switching from IgM to IgG, IgA or IgE IgD is an exception (no switch region) L V DJ Cµ Cδ C γ3 C γ1 C α1 C γ2 C γ4 C α2 Cε 5' 3' Sµ S γ3 S γ1 S α1 S γ2 S γ4 S α2 Sε Sequential Class Switch L V DJ C γ4 Cε 5' 3' Sµ γ4 Sε L V DJ Cε 5' 3' Sµ γ4ε
Effector Functions Typically Inflammatory Reactions Complement Fixation Complement Activation Binding to Fc Receptors Basophils Monocytes Platelets
IgM First Ig to be produced Can exist as a monomer or a pentamer Excellent for agglutination, complement activation
IgG bound to FcRn Igs can function as the antigen binding component for many receptors and signal transduction proteins on many types of cells B cells, membrane Ig binds Igα and Igβ Granulocytes, secretory Ig binds Fc receptors
Effector functions of human isotypes Igs can kill in one of two ways: ADCC (antibody dependent cell-mediated cytotoxicity, Fc receptor driven Complement lysis www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
IgA Major Ig class in secretions, critical to mucosal immunity Can exist as a monomer or dimer Transported by means of secretory component
Nature 406, 259-266 (2000) IgE bound to FcεRI
Average Serum Immunoglobulin Con (mg/dl) Average Serum Immunoglobulin Con (mg/dl) ncentration ncentration Ontogeny of Serum Ig IgM, IgG, IgA IgG Subclasses 1300 1100 900 700 Maternal IgG IgG 900 700 500 IgG 1 500 300 300 250 Child's IgG IgA 250 200 IgG 2 200 150 100 IgM 150 100 IgG 3 50 25 0 0 2 4 6 8 2 4 6 8 10 12 2 4 6 8 10 15 25 35 45 55 65 75 85 Months Months Years Birth Age 50 25 0 IgG 4 2 4 6 8 10 12 2 4 6 8 10 15 25 35 45 55 65 75 85 Months Years